Studies have been conducted to optimize the use of recombinant vaccines to enhance immune responses and anti-tumor activity in preclinical models, to better understand the underlying principles and mechanisms in immunologic tolerance to tumor antigens, and to develop novel strategies for the more effective use of cancer vaccines that can be translated to science-driven clinical studies. The development of safe, novel adjuvants is necessary to maximize the efficacy of new and/or available vaccines. Chitosan is a non-toxic, biocompatible, biodegradable, natural polysaccharide derived from the exoskeletons of crustaceans and insects. Chitosans biodegradability, immunological activity and high viscosity make it an excellent candidate as a depot/adjuvant for parenteral vaccination. To this end, we explored chitosan solution as an adjuvant for subcutaneous vaccination of mice with a model protein antigen. We found that chitosan enhanced antigen-specific antibody titers over 5-fold and antigen-specific splenic CD4+ proliferation over 6-fold. Strong increases in antibody titers together with robust delayed-type hypersensitivity (DTH) responses revealed that chitosan induced both humoral and cell-mediated immune responses. When compared with traditional vaccine adjuvants, chitosan was equipotent to incomplete Freunds adjuvant (IFA) and superior to aluminum hydroxide. Mechanistic studies revealed that chitosan exhibited at least two characteristics that may allow it to function as an immune adjuvant. First, the viscous chitosan solution created an antigen depot. More specifically, less than 3% of a protein antigen, when delivered in saline, remained at the injection site after 8 hours. However, more than 60% of a protein antigen delivered in chitosan remained at the injection site for 7 days. Second, chitosan induced a transient 67% cellular expansion in draining lymph nodes. The expansion peaked between 14 and 21 days after chitosan injection and diminished as the polysaccharide was degraded. These mechanistic studies, taken together with the enhancement of a vaccine response, demonstrate that chitosan is a promising and safe platform for parenteral vaccine delivery. Only one adjuvant (alum) has been approved by the FDA for use with vaccines. In addition to potential safety issues, its principal use is for the enhancement of vaccine-mediated antibody responses. The studies reported here demonstrate that chitosan is extremely safe and extremely effective in enhancing vaccine-mediated antibody and T cell responses. These studies thus have important implications for immunotherapy and immunoprevention protocols for a range of human cancers. CD4+CD25+Foxp3+ regulatory T (Treg) cells have been implicated in the lack of effective anti-tumor immunity. Denileukin diftitox (DAB389IL-2, ONTAK), a fusion protein of IL-2 and diphtheria toxin, provides a means of targeting Treg cells. This study was designed to determine how to optimize vaccine efficacy with the use of an immunotoxin to reduce regulatory T cells. In this study, we examined in a preclinical model (a) the effect of ONTAK on the deletion of Treg cells in various lymphoid compartments and (b) the dose scheduling of ONTAK in combination with a recombinant poxviral vaccine to enhance antigen-specific immune responses. Treg cells in spleen, peripheral blood, and bone marrow of normal C57BL/6 mice were variously reduced after a single intraperitoneal injection of ONTAK; the reduction was evident within 24 hours and lasted approximately 10 days. Injection of ONTAK 1 day prior to vaccination enhanced antigen-specific T-cell responses above levels induced by vaccination alone. These studies demonstrate for the first time in a murine model (a) the differential effects of ONTAK on Tregs in different cellular compartments, (b) the advantage of combining ONTAK with a vaccine to enhance antigen-specific T-cell immune responses, (c) the lack of inhibition by ONTAK of host immune responses directed against a live viral vector, and (d) the importance of dose scheduling of ONTAK when employed in combination with a vaccine. These studies have a direct implication in the design of clinical trials for a range of human cancers employing vaccines and an agent that can enhance its efficacy. Other accomplishments of this project include: 1. Enhanced levels of costimulation lead to reduced effector/memory CD8+ T cell functionality. Mostbck S, Vidal S, Schlom J, and Sabzevari H. J. Immunol. (in press) 2. The IL-2 immunotoxin denileukin diftitox reduces regulatory T cells and enhances vaccine-mediated T-cell immunity. Litzinger MT, Fernando R, Curiel TJ, Grosenbach DW, Schlom J, and Palena C. Blood (in press) 3. Chitosan solution enhances both humoral and cell-mediated immune responses to subcutaneous vaccination. Zaharoff DA, Rogers CJ, Hance KW, Schlom J, and Greiner JW. Vaccine 25:2085-2094, 2007. 4. Acquisition of antigen presentasomes (APS), an MHC/costimulatory complex, is a checkpoint of memory T cell homeostasis. Mostbck S, Catalfamo M, Tagaya Y, Schlom J and Sabzevari H. Blood 109:2488-2495, 2007. 5. Pharmacokinetics and tumor localization of 111In-labeled HuCC49&#916;CH2 in BALB/c mice and athymic murine colon carcinoma xenograft. Chinn PC, Morena RA, Santoro DA, Kazules T, Kashmiri SVS, Schlom J, Hanna N, and Braslawsky G.Cancer Biother. Radiopharm. 21:106-116, 2006. 6. Vaccines with enhanced costimulation maintain high avidity memory CTL. Yang S, Hodge JW, Grosenbach DW, and Schlom J. J. Immunol. 175:3715-3723, 2005. 7. Intratumoral delivery of vector mediated IL-2 in combination with vaccine results in enhanced T-cell avidity and anti-tumor activity. Kudo-Saito C, Garnett CT, Wansley EK, Schlom J, and Hodge JW. Cancer Immunol. Immunother. (in press) [e-pub] 8. Synergistic antitumor activity of immune strategies combined with radiation. Sharp HJ, Wansley EK, Garnett CT, Chakraborty M, Camphausen K, Schlom J, and Hodge JW. Front. Biosci. (in press) 9. The combined activation of positive costimulatory signals with modulation of a negative costimulatory signal for the enhancement of vaccine mediated T-cell responses. Chakraborty M, Schlom J, and Hodge JW. Cancer Immunol. Immunother. 56:1471-1484, 2007. 10. Combination therapy of an orthotopic renal cell carcinoma model employing intratumoral vector-mediated costimulation and systemic IL-2. Kudo-Saito C, Wansley EK, Gruys ME, Wiltrout R, Schlom J and Hodge J. Clin. Cancer Res. 13:1936-1946, 2007. 11. 4-1BB ligand enhances tumor-specific immunity of poxvirus vaccines. Kudo-Saito C, Hodge JW, Kwak H, Schlom J, and Kaufman HL. Vaccine 24:4975-4986, 2006. 12. Radiation modulates the peptide repertoire, enhances MHC class I expression and induces successful anti-tumor immunotherapy. Reits EA, Hodge JW, Herberts CA, Groothuis TA, Chakraborty M, Wansley E, Camphausen K, Luiten RM, de Ru AH, Neijssen J, Griekspoor A, Mesman E, Verreck FA, Spits H, Schlom J, van Veelen P, and Neefjes JJ. J. Exp. Med. 203:1259-1271, 2006